The increased atherosclerosis in the presence of high plasma HDL levels in LCAT transgenic (Tg) mice has been proposed to be due at least in part to dysfunctional HDL. To determine the functionality of LCAT Tg HDL on cellular cholesterol (chol) metabolism and efflux we incubated control and LCAT Tg HDL with cultured human fibroblasts previously radiolabeled with [14C]FC-PC liposomes to label chol in the plasma membrane (PM), [14C] and [3H]oleate for the chol ester storage pool and [14C]acetate to radiolabel newly synthesized chol in the smooth endoplasmic reticulum (SER). In addition, mass determination of cellular chol in human macrophages after efflux with HDL was used to determine net chol efflux. We found that there was a 40-50% decrease (P<0.01) in efflux of newly synthesized chol in the presence of LCAT Tg HDL compared to control HDL. Also the ability of LCAT Tg HDL to promote net chol efflux in macrophages was impaired. In addition, LCAT Tg HDL did not inhibit chol esterification to the same extent (30% reduction) that was reached when cells were incubated with control HDL (50% reduction)(P<0.01). In summary: 1) When compared to control HDL, LCAT Tg HDL has decreased ability to promote efflux of newly synthesized chol and is less effective in suppressing ACAT mediated esterification of FC. 2) Thus, LCAT Tg HDL is dysfunctional and not effective in cholesterol efflux resulting in decreased reverse chol transport which may be an important mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels. - Human Subjects